Cellulose is an unbranched polymer of glucose linked by β(1→4)-glycosidic bonds. Cellulose chains can interact with each other via hydrogen bonding to form a crystalline solid of high mechanical strength and chemical stability. The cellulose chains must be depolymerized into glucose and short oligosaccharides before organisms, such as the fermenting microbes used in ethanol production, can use them as metabolic fuel. Cellulase enzymes catalyze the hydrolysis of the cellulose (hydrolysis of β-1,4-D-glucan linkages) in the feedstock into products such as glucose, cellobiose, and other cellooligosaccharides. Cellulase is a generic term denoting a multienzyme mixture comprising exo-acting cellobiohydrolases (CBHs), endoglucanases (EGs) and β-glucosidases (βG) that can be produced by a number of plants and microorganisms. Enzymes in the cellulase of Trichoderma reesei include CBH1 (more generally, Cel7A), CBH2 (Cel6A), EG1 (Cel7B), EG2 (Cel5), EG3 (Cel12), EG4 (Cel61A), EG5 (Cel45A), EG6 (Cel74A), Cip1, Cip2, β-glucosidases (including, e.g., Cel3A), acetyl xylan esterase, β-mannanase, and swollenin.
Cellulase enzymes work synergistically to hydrolyze cellulose to glucose. CBH1 and CBH2 act on opposing ends of cellulose chains (Barr et al., 1996), while the endoglucanases act at internal locations in the cellulose. The primary product of these enzymes is cellobiose, which is further hydrolyzed to glucose by one or more β-glucosidases.
The kinetics of the enzymatic hydrolysis of insoluble cellulosic substrates by cellulases do not follow simple Michaelis-Menten behaviour (Zhang et al., 1999). Specifically, increasing the dosage of cellulase in a hydrolysis reaction does not provide a linearly dependent increase in the amount of glucose produced in a given time. There is also a significant decrease in the rate of reaction as cellulose hydrolysis proceeds (Tolan, 2002). Several explanations have been proposed to explain the decline in the reaction rate; the major hypotheses include substrate heterogeneity (Nidetsky and Steiner, 1993; Zhang et al., 1999), enzyme inactivation (Caminal et al., 1985; Converse et al., 1988; Gusakov and Sinitsyn, 1992; Eriksson et al., 2002), and product inhibition (Lee and Fan, 1983; Caminal et al., 1985; Holtzapple et al., 1990; Gusakov and Sinitsyn, 1992; Eriksson et al., 2002; Gruno et al., 2004).
Inhibition of enzymes by the products of the reactions they catalyze has long been recognized; the phenomenon was known to Henri, Michaelis, and Menten, all pioneers in the field of enzymology (Frieden and Walter, 1963). The nature of product inhibition may be competitive, as product competes with substrate to form the same interactions with the enzyme, but other forms of inhibition are possible. Indeed, due to the insoluble nature of cellulose and the challenges it poses as a substrate in kinetic studies, there have been many conflicting reports as to the nature of inhibition in the cellulase system (Holtzapple et al., 1990, and references therein). The cellobiohydrolases are subject to inhibition by their direct product, cellobiose, and to a lesser degree by the glucose produced by the further hydrolysis of the cellobiose by β-glucosidase. One technique for reducing cellulase inhibition is to increase the amount of β-glucosidase in the system (U.S. Pat. No. 6,015,703), as cellobiose is more inhibitory to cellulases than glucose (Holtzapple et al., 1990; Teleman et al., 1995). Inhibition can be mitigated by altering the primary sequence of the protein using DNA mutagenesis guided by rational design or applied randomly. For example, rational design was used to target the Y245 residue in Cel5A, an endoglucanase, for mutagenesis, which resulted in an increase in its cellobiose inhibition constant (U.S. Publication No. 2003/0054535).
There are relatively few reports of engineering Cel6A (also known as cellobiohydrolase II or CBH2), a major cellobiohydrolase of the T. reesei (also known as Hypocrea jecorina) cellulase system, for desirable properties. St-Pierre et al. (U.S. Publication No. 2008/0076152) have shown that substitution of the naturally occurring amino acids at the equivalent of positions 231, 305, 410 and 413 in the T. reesei Cel6A sequence to serine or threonine (positions 231 and 305), glutamine or asparagines (position 410) or proline (position 413) increase thermostability, thermophilicity and/or alkalophilicity of a Family 6 cellulase. Wohlfahrt et al. have enhanced the stability of the protein by forming amide-carboxylate pairs through mutagenesis at residues E107, D170 and D366 (U.S. Publication No. 2004/0152872). Rational design was also applied to a related cellobiohydrolase, Cel6B from Thermobifida fusca, to relieve inhibition by cellobiose (Zhang et al., 2000). Mutations at Cel6B residues equivalent to W269, H266, and E399 in the Cel6A sequence were shown to reduce cellobiose inhibition, but at a significant cost to activity on crystalline cellulose. Another approach, based on the consensus sequence derived from an alignment of Cel6A sequences from several species (U.S. Publication No. 2006/0205042), identified 38 amino acids associated with improved thermostability (specifically: V94, P98, G118, M120, M134, T142, M145, T148, T154, L179, Q204, V206, I212, L215, G231, T232, V250, Q276, N285, S291, G308, T312, S316, V323, N325, I333, G334, S343, T349, G360, S380, A381, S390, F411, S413, A416, Q426 and A429). The authors speculate that these mutations may also affect product inhibition and/or enzyme processivity, but offer no data or specific hypotheses based on modeling to associate changes in these properties with the claimed residues. The consensus approach is designed to generate protein variants with improved thermodynamic stability (Steipe, 2004) and it does not provide predictive power for the improvement of any other biochemical property.
Although cellulase compositions have been described previously, there remains a need for new and improved compositions for use in the conversion of cellulose into fermentable sugars and for related fields of cellulosic material processing such as pulp and paper, textiles and animal feeds. Cellulases with improved performance decrease the cost of the processes and typically offer substantial environmental benefits when compared to the equivalent chemical and/or physical processes. For example, the production of fuel ethanol from cellulose delivers substantial environmental and economic benefits. When compared to gasoline, using ethanol as a fuel significantly reduces net carbon emissions by fixing the carbon dioxide released during combustion back into the biomass grown as feedstock for further ethanol production. Using agricultural biomass as feedstock can also stimulate rural economies and reduce dependence on foreign petroleum. Producing ethanol from cellulose rather than starch, as for corn ethanol, or sugar has the additional benefit of avoiding competition with the production of foodstuffs for humans and animals. The US Departments of Agriculture and Energy estimate that 30% of transportation fuel use in America, the largest petroleum market in the world, could be displaced by using cellulosic fuel without affecting food harvests (Perlack et al., 2005). Additionally, due to the low energy input required to generate cellulosic biomass, it has been estimated that the use of cellulose ethanol reduces net greenhouse gas production by 88% when compared to gasoline whereas using corn ethanol produces a decrease of only 18% (Farrell et al., 2006).